The long-term objective of this project is to learn how ACTH, angiotensin and K plus stiumlate steroidogenesis in Y-1 adrenal tumor cells, adrenal glomerulosa cells and adrenal fasicilata cells. These cell types are chosen because they provide model systems for studying cyclic AMP independent steroidogenesis. The studies proposed here are designed to measure phosphoproteins produced in response to varying concentrations of ACTH, angiotesin and K plus using a newly devised two-dimensional electrophoresis - double labelling procedure. Intact cells will be incubated with 32Pi or 33Pi to permit labelling of the phosphoproteins. Cells labelled with 33Pi will be treated with ACTH, ACTH partial agonist analogs, angiotensin, K plus, 8-Br-cyclic AMP or cholera toxin whereas those grown in 32Pi will serve as controls. Stimulated and control cells will be pooled and lysed in a buffer containing urea and MgCl2. The phosphoproteins in the mixture will be separated by two-dimensional gel electrophoresis and measured by differential autoradiography. Changes in the 32P/33P content of individual phosphoprotein will be correlated with the degree of stimulation of steroidogenesis. Cyclic AMP-dependent and cyclic AMP-independent protein kinases will be purified and used to induce phosphoprotein labelling in cell homogenates. The labelling pattern produced will be compared with that induced by hormonal stimulation to ascertain if the actions of cyclic AMP and cyclic AMP-dependent protein kinases are sufficient to account for phosphorylation seen in response to hormonal stimulation. This procedure should clarify the role of cyclic AMP in mediating steroidogenesis. In addition, 3 specific phosphoproteins will be isolated and purified following hormonal stimulation. These are cholesteryl ester hydrolase, phosphorylase, and tubulin. The first two are believed to be phosphorylated and activated during hormone induced steroidogenesis. Phosphorylation of tubulin may be responsible for the rounding of Y-1 cells in culture after ACTH and/or cyclic AMP treatment.